DNA encoding ubiquitin conjugating enzymes

ABSTRACT

The invention relates to methods for determining a predisposition for and diagnosing the existence of a degenerative disease or a cancer and also products and processes for treating and obtaining treatments for such a degenerative disease or a cancer. The invention has particular application in the use of information concerning the elucidation of DNA and amino acid sequence structure relating to human and mouse ubiquitin conjugating enzymes.

This application is a 371 of PCT/GB95/00707, filed on Mar. 29, 1995.

The invention relates to a method for determining a predisposition forand diagnosing the existence of a degenerative disease, particularly,but not exclusively, Alzheimer's Disease (AD), and also products andprocesses for treating and obtaining treatments for such a degenerativedisease. The single inventive concept also relates to products andprocesses for treating and obtaining treatments for Down's syndrome. Inaddition, the single inventive concept also relates to a method fordetermining a predisposition for and diagnosing the existence of cancer,particularly, but not exclusively, papilloma virus induced cancers, andalso products and processes for treating and obtaining treatments forsuch cancers.

In the following application the term degenerative disease will be takento mean a disease characterised by an aberration in the ubiquitinationpathway and/or intermediate filament inclusion bodies of a diverse typesuch as those found in Parkinson's Disease, Pick's Disease, Alzheimer'sDisease, as well as Rosenthal fibres in Cerebellar Astrocytomas,Cytoplasmic bodies in muscle and Mallory bodies in Alcoholic LiverDisease.

The degenerative neurological disease, Alzheimer's Disease, is one ofthe most challenging medical problems of our day. AD is a chronic,progressive, irreversible and fatal age-related dementia. It hasreceived increased attention in recent years because of currentdemographic data, which data shows a continuously increasing proportionof elderly people within the population. The disease appears to bespecific for humans and aged primates only and its causes are largeiyunknown. The disease presents a major public health problem in thatcaring for demented patients is particularly demanding on health careresources.

Studies of the disease are complicated by the fact that a reliablediagnosis depends on analysis of brain tissue, suicIh diagnosis usuallybeing conducted post-mortem and being of no use to the patientconcerned. Putative clinical diagnosis is made principally bv exclusionof other causes of dementia--a largely unsatisfactory situation.

Studies are further hindered by the fact that useful animal or cellmodels are not yet available for investigation. Hence the development oftherapeutic agents for treating the disease is not vet possible.

An understanding of the disease is further complicated by the fact thatin some cases AD is familial, whereas in other cases it is sporadic.However, in all instances post-mortem diagnosis is determined by thedistinctive neuropathological features of the disease. These featurescomprise two conspicuous types of deposits; amyloid plaques andneurofibrillary tangles (NFTs). The plaques consist of aggregates of apeptide derived from amyloid precursor transmembrane protein (APP). Theplaques are generated by abnormal protein metabolism. NFTs and theirconstituents, the paired helical filaments (PHFs) consist largely of amicrotubular-associated protein, known as tau, in various abnormalstates of phosphorylation. It is known that PHFs are very highlyinsoluble and this makes their analysis and characterisation extremelydifficult. In addition, it is known that NFTs are associated with theprotein ubiquitin; however, the significance of this association isunknown. That ubiquitin is a component of paired helical filaments wasfirst disclosed in 1987. It is also known that ubiquitin is a commonfactor in intermediate filament inclusion bodies of a diverse type inman such as those found in Parkinson's Disease, Pick's Disease,Alzheimer's Disease as well as Rosenthal fibres in CerebellarAstrocytomas, cytoplasmic bodies in muscle and Mallory bodies inAlcoholic Liver Disease. However, the significance of these associationsis unknown.

It is also interesting to note that the pathology of AD is also found invictims of Down's syndrome. The significance of this will be describedin greater detail hereinafter.

It is, however, known that the ubiquitination pathway functions totarget cellular proteins for degradation. The pathway is thought tooperate in all cell types and is necessary for cell viability.Ubiquitination is particularly important in the control of:proliferation and differentiation; DNA repair; heat shock response; andorganelle formation. A functional de-ubiquitination system is alsonecessary for cell viability. Short half-life proteins such as thosewhich control progress through the cell cycle are targeted fordegradation by ubiquitination. Abnormal and mutant proteins areprocessed in a similar way. It is of note that many proteins areresistant to proteolytic digestion in the absence of ubiquitination.Despite the possible involvement of ubiquitin in degenerative diseasepathology and its importance in protein processing, there is no priorart describing the role of ubiquitin associated enzymes and, inparticular, ubiquitin conjugating enzymes in degenerative diseases andin particular in AD.

Moreover, four human ubiquitin conjugating enzymes have been discoveredbut it is of note that none of these enzymes maps to a chromosome regionhitherto implicated in degenerative diseases and in particular inAlzheimer's Disease (5, 6, 7).

The ubiquitination pathway involves a cyclical four step process. Theprocess includes a number of enzymes such as: ubiquitin activatingenzymes (E1), ubiquitin conjugating enzymes (E2 or UBC),ubiquitin-protein ligases (E3) and proteosomes but, as mentioned, thereis no suggestion in the prior art that any of these enzymes has a roleto play in degenerative disease and in particular in Alzheimer'sDisease.

In order that the invention may be understood the ubiquitination pathwaywill be described in greater detail.

In the following description enzymes E1, E2 (or UBC) and E3 prefixedwith the letter H, for example HUBC4, represent the human version of therelevant enzyme such as human ubiquitin conjugating enzyme and enzymesE1, E2 (or UBC) and E3 without this prefix for example UBC4 representthe yeast version of the relevant enzyme such as yeast ubiquitinconjugating enzyme.

It is known that the ubiquitination pathway has a major role to play, atleast, in the selective degradation of normal and short-lived proteinssuch as those that control progress through the cell cycle for eg p53(8). The pathway firstly involves activation of ubiquitin by the enzymeE1 in at ATP-dependent manner. Activation involves the formation of athioester between the active cysteine residue of E1 and the C-terminalglycine of ubiquitin. Once activated, the ubiquitin is transferred to acysteine residue of a ubiquitin-conjugating enzyme (such as UBC4 orHUBC4). The ubiquitin-conjugating enzyme then catalyses the formation ofan isopeptide bond between the C-terminal glycine of ubiquitin and theE-amino group of a lysine residue on a target protein. This is broughtabout by E3 ubiquitin ligases specifically binding to target proteinsthat are not otherwise recognised by E2's. In addition, ubiquitin alsobecomes conjugated to itself via a lysine residue at position 48 ofubiquitin resulting in the formation of multiubiquitin chains.Multiujbiquinated proteins serve as targets that are recognised anddegraded by an ATP-dependent protease complex.

The ubiquitin-conjugating enzymes or E2's comprise a family of proteinscharacterised by a highly conserved catalytic site. In yeast at least 10different E2's have been identified. A number of these E2's, such asUBC1, UBC4 and UBC5 play an important role in the specific targetedbreakdown of abnormal and short-lived proteins.

Another fraction of E2's catalyse the transfer of ubiquitin to smallproteins, such as histones, in a reaction that does not require E3.These reactions result in monoubiquitin derivatives that do not serve asproteolysis intermediates.

It is of note that in yeast the ubiquitin conjugating enzymes (E2) UBC4and UBC5 show a 92% protein sequence homology and mutation in both thesegenes is required for a substantial reduction in growth rates. Inaddition, over expression of UBC1 complements UBC4 and UBC5 mutations inyeast. It would therefore seem that these enzymes have significantstructural and functional similarities and in view of these facts weconsider that both the functionally equivalent human ubiquitinconjugating enzymes HUBC5 and HUBC1 have likewise a significant role toplay in the diagnosis and treatment of the diseases described herein.For example, it may be that a mutation or polymorphism in HUBC4 will becomplemented by a mutation or polymorphism and/or over expression ofHUBC5 and/or similarly, a mutation or polymorphism and/or overexpression of HUBC1.

It may be that an aberration in any one of the human ubiquitinconjugating enzymes results in the diseases described herein.

The ubiquitination pathway also has relevance in the control of cellgrowth or cell division and thus abberations in this control aretypically characterised by cancerous growths and particularly papillomavirus induced cancerous growths. This has been realised as a result ofthe following information.

It has recently been reported that ubiquitin-conjugating enzymes areinvolved in ubiquitination of p53 (1,2,3).

p53 is involved in the cell cycle and more specifically it is a G₁ -Scheckpoint protein. During the cell cycle the cell enters a number ofdefined stages termed G₁, where the cell prepares for replication, S,where cellular DNA is replicated, G₂ an interval prior to cellseparation, and M, where the cell divides. During G₁, p53 essentiallychecks the integrity of the cell and if mutations in the DNA areidentified p53 prevents commencement of S phase and so prevents cellreplication. In this way, p53 ensures that abberations in cellular DNAare not propagated by cell division. It therefore follows that p53 is animportant cellular protein, indeed, it has been termed a tumoursuppressor protein. Multi-ubiquitination of this protein thereforetargets it for destruction and removes from the cell an importantcontrol agent.

Since the agent responsible for ubiquitination of p53 is a ubiquitinconjugating enzyme it follows that mutations in this enzyme whichdeleteriously affect cellular levels of p53 will have a role to play intumour formation.

In addition, over expression of the human ubiquitin-conjugating enzymeactive against p53, will have deleterious consequences.

Abberations in p53 control are known to be associated with cancerscaused by the human papilloma virus (HPV) and specifically types 16 and18 which appear to stimulate the ubiquitin-dependent degradation of thep53 tumour suppressor protein.

HPV-16 and HPV-18 are typically associated with malignant legions suchas cervical cancer and head and neck cancer. These two HPV types arereferred to as high risk HPV's as opposed to low-risk HPV's which aregenerally associated with benign lesions. Both of the high-risk HPV'sencode proteins, termed E6 and E7, which bind to cellular regulatoryproteins. Specifically E6 binds to a cellular protein of 100KDa, termedE6-associated protein (E6-AP). This E6-E6-AP complex binds p53 and soenables a ubiquitin-conjugating enzyme to ubiquitinate p53. It wouldtherefore seem that E6 when bound to the E6-associated protein, acts asa ubiquitin protein ligase or E3.

It would therefore seem that the human papilloma virus is adapted tomake use of the cellular ubiquitination pathway and so overcome the G₁-S checkpoint which prevents replication of aberrant DNA.

It has recently been reported that it is possible to ubiquitinate p53 inthe absence of E6. Indeed, a novel rabbit ubiquitin conjugating protein,designated E2-F1, has been described in the ubiquitination of non-"N-endrule" substrates such as glyceraldehyde-3-phosphate dehydrogenase andp53. (4)

We consider therefore that a HUBC4 is recruited by the E6-E6-AP/p53complex in HPV 16 or 18 affected cells with the result that p53 isubiquitinated more rapidly than in normal cells and thus is more rapidlydegraded.

This results in the infected cell behaving as if it has lost p53function. Mutations in the p53 gene resulting in the loss of functionalp53 are well known to result in a malignant phenotype. Thus we believethat agents which either interfere with the interaction between E6-E6-APand a HUBC4 or with the interaction between E6-E6-A6/HUBC4 complex andp53 would function as treatments for HPV induced cancers for example,cervical cancers. We also believe that antagonists of the HUBC4 enzymewill also function as treatments for HPV infected cells and HPV inducedcancers.

We also consider that abberations, polymorphisms or mutations in theHUBC4 gene may have a part to play in the inappropriate or excessiveubiquitination of p53 and thus in the untimely removal of an importantmeans of cellular control and the development of unregulated celldivision.

In some instances, a degenerative disease such as for exampleAlzheimer's Disease is familial. It is therefore logical to suppose thatthe gene or genes relating to these familial diseases such asAlzheimer's Disease are located on particular chromosomes. To this end,a number of genetic investigations have been undertaken. Investigationsrelating to AD will now be summarized.

It has been found that familial AD appears to segregate as an autosomaldominant trait. In some families mutations in the amyloid precursorprotein (APP) have been described. For example mutations at amino acid670, 671, 692, 693 or 771 of this protein have sometimes been found tosegregate with AD in families. The APP gene is located on humanchromosome 21 at location 21q21.3 and was initially seen as aparticularly attractive candidate gene for giving rise to Alzheimer'sDisease because patients with Down's syndrome (chromosome 21 trisomydysfunction) often show the pathological features of AD when theirbrains are examined at autopsy. However, the proportion of familial ADcases associated with mutations in the APP gene on chromosome 21 issmall. In general, the APP mutations described to date involveconservative amino acid changes which a priori would not be predicted toexert a major effect on the behaviour of the resultant protein.

Other workers have described pedigrees segregating senile onset familialAlzheimer's Disease and suggested an association with genetic markers onchromosome 19q. However, once again the number of families falling intothis category is relatively small.

A large number of genetic studies have recently suggested that a generesponsible for familial AD maps to human chromosome 14 and inparticular 14q24.3. Indeed, there has also been some suggestion that thegene on human chromosome 14 may in fact also be playing a role in thefamilies where linkage to chromosome 21 is suspected. The individualswho have established this genetic linkage have speculated about genesknown to lie on human chromosome 14 and their potential involvement inthe pathology of AD. Such genes include the protease inhibitors AACT,PI, the protease Cathepsin G and TGFβ3. In addition, the transcriptionfactor c-FOS maps to the 14q24 region of chromosome 14. There has beenspeculation on the possible role of c-FOS because this transcriptionfactor may be involved in the transcriptional regulation of the APPgene. However, the significance of this in Alzheimer's Disease remainsunestablished. Furthermore, the 70 KDa heat shock protein HSPA2 alsomaps to the 14q24 region of chromosome 14. The product of this gene is amolecular chaperone potentially involved in protein folding and assemblyand could thus act theoretically in APP processing so that mutationswould possibly lead to later amyloid deposition. However, once againthere is no evidence to suggest that this hypothesis is true in AD.

In conclusion, the prior art is confusing in that it suggests that thereare any number of genetic influences on the development of familial AD.What is clear, however, is that to date, individuals skilled in the arthave not been able to quickly isolate the genes involved in AD andparticularly familial AD.

In addition, the genes involved in the other herein referred todegenerative diseases and cancers have also not been isolated.

Our investigations have led us to identify a surprising number of geneslocated on a number of chromosomes but coding for almost identical, orat least very similar proteins, which proteins would seem to beubiquitin conjugating enzymes. One gene maps to human chromosome 14 atlocation 14q24.3. This gene has never previously been described. We havecalled this gene and the protein it encodes human ubiquitin conjugatingenzyme 4 (HUBC4, and more specifically HUBC4a). Our investigations havealso lead us to identify other genes that code for human ubiquitinconjugating enzymes which genes map to: human chromosome 22 at location22 q12-13, human chromosome 12 at location 12 p and human chromosome 19and human chromosome 13. These genes have never previously beendescribed. Surprisingly all these genes encode for human ubiquitinationenzymes specifically enzymes identical to or variants of HUBC4a. We havecalled these genes HUBC4b, HUBC4c and HUBC4d and HUBC4e, respectively.

Experiments have shown that a rabbit protein is active as a ubiquitincarrier protein in rabbit reticulocyte lysates (4). Partial sequencingof tryptic digestion fragments from this protein are homologous tofragments of the protein encoded by our HUBC4b gene. Thus it can besuggested that a rabbit protein related to our HUBC4b gene product has apart to play in the cellular ubiquitination pathway in that species andso it is likely that the HUBC4b gene product and the variants thereofhereindescribed equally have a part to play in the human cellularubiquitination pathway.

The genes which we have therefore successfully identified are likely toform part of the cellular ubiquitination pathway and are typicallylikely to be involved in targeting cellular proteins for degradation.Since it is known that proteins are resistant to proteolytic digestionin the absence of ubiquitination it is not surprising that mutations inthe genes encoding the enzymes which are part of the ubiquitinationprocess can lead to an increase in protein half-life within the cell andcorresponding deleterious consequences such as an inability to completethe normal cell cycle. Indeed, we believe that mutations in either theubiquitination pathway or the target protein for ubiquitination in humantissues result in pathological consequences. Mutations in theubiquitination enzymes and in particular in the herein described classof HUBC4 enzymes lead to decreased or enhanced protein ubiquitination.The former variant may affect cell proliferation and may also beinvolved in generation of intermediate filament inclusion bodies ofdifferent types in several major human diseases and in particular inAlzheimer's Disease. The latter variant may affect cell growth controland so lead to cancerous growths.

Having identified the HUBC4 class of genes we have now provided a humancDNA, named HUBC4b, which encodes a protein of 154 amino acid residueshaving a molecular weight of 17.9 KDa and that demonstrates a 55%homology in primary sequence, and also displays a similar hydrophobicityprofile, to that of the yeast E2 gene UBC4. The key cysteine residuethought to become involved in thioester bond formation and specificadjacent amino acids are conserved. It is of note that there is no priorart teaching that a human homologue of the yeast UBC4 gene orcorresponding protein might have any role or relevance in degenerativediseases and in particular in AD. Indeed, human homologues of yeast UBCgenes have been said to have a role to play in DNA repair, DNAreplication, G1-S cell cycle progression and G2 check point progression.Hence this prior art teaching tends to lead away from the hypothesisthat ubiquitin conjugating enzymes, as a class, might have an importantrole to play in AD or other diseases and makes our suggestion that theHUBC4 class of genes is involved in degenerative diseases, and inparticular in AD, all the more unexpected.

The cDNA for any of the HUBC4 class of genes or any single one of thesegenes including genetic control elements and introns may be used forgene therapy approaches using nucleic acid delivery vectors (viruses,liposomes, etc) and methods for achieving tissue-specific expressionwhich are known to the skilled man. Provision of a cDNA clone from anyof the aforementioned genes and the genomic DNA sequence as well asYeast Artificial Chromosomes containing any one of the entire genes willallow the sequence of introns at the intron-exon boundaries to bedetermined by methods well-known to the skilled man. The availability ofthis data allows a number of well-known technologies for the detectionof mutations to be applied, for example, but not limited to, SSCP (5).Thus we provide DNA sequence data which will allow the identification ofpolymorphisms and/or mutations in the class of human HUBC4 genes and soaid in the diagnosis of degenerative diseases such as familial AD and/orsporadic AD and also in the diagnosis of a vulnerability to, at least,human papilloma virus induced cancer. The sequences defined for thefirst time herein will allow the development of diagnostic tests formutations either at the DNA or RNA level again using methods known tothe skilled man such as PCR for example using the "ARMS" technique (6).

In summary therefore, our invention concerns the identification of geneswhich we call HUBC4a, HUBC4b, HUBC4c, HUBC4d and HUBC4e, mutations inwhich we believe are responsible, at least, for the degenerativediseases described herein and cancers in particular papilloma virusinduced cancers.

Our invention is consistent with observations made in the prior art suchas: the fact that familial AD is thought to involve a gene on chromosome14 at location 14q24.3 and one of our genes maps to this location; thatmutations at this locus are consistently found to segregate withAlzheimer's Disease in pedigrees; and that ubiquitin is well-known to beinvolved in protein degradation and that therefore ubiquitin conjugatingenzyme anomalies may result in the inappropriate accumulation of proteindeposits and disease and/or a lack of cell growth control.

We speculate that mutant forms of ubiquitin conjugating enzymes showvariable activity when functioning as ubiquitin conjugating enzymes.Over many years this leads to alterations in the rates of proteinprocessing particularly in the CNS and eventually leads to the build tipof intermediate inclusion bodies such as NFTs and/or amyloid plaques.

We also provide the sequence of cDNA for a ubiquitin conjugating enzymein the mouse (mUBC4b). The availability of this sequence will allowgenomic clones for the corresponding mouse ubiquitin conjugating enzymeto be isolated, again using well-known methods. The availability of suchmouse sequence will allow transgenic mice to be prepared usingestablished technologies where the function of this enzyme is eitherdestroyed by gene knockout or modified by the introduction of specificmutations. In this fashion, mouse animal models will be created whichdevelop symptoms characteristic of degenerative diseases such as ADhomologous to the condition in man; and/or models will be created whichare susceptible to cancers and in particular papilloma virus inducedcancers. Any compound which delays or prevents the appearance of thepathological features of these diseases in such transgenic animals islikely to have therapeutic benefit in man.

We also provide herein the predicted protein sequences of humanubiquitin conjugating enzymes HUBC4a, HUBC4b and HUBC4c from humanchromosome 14, 14q24.3; human chromosome 22q; and human chromosome 12p,respectively. These proteins themselves may have therapeutic uses.

Of particular value are monoclonal or polyclonal antibodies raised usingstandard techniques either to the whole protein, to epitopes orfragments thereof, to truncated variants thereof, to splicing variantsthereof and to protein variants displaying mutation and amino acidchanges. Such variants will be identified as described above by, forexample but not limited to SSCP analysis (5) of the DNA from affectedpatients. Antibodies such as these will have utility in establishing thediagnosis of degenerative diseases such as, but not limited to, AD inpost-mortem samples and may facilitate analysis of neurologicalbiopsies.

In addition, these antibodies may be usefully employed to block selectedHUBC4 enzymes in order to prevent or at least mitigate p53 degradation.

We also consider that our invention has important implications for thetreatment of Down's syndrome. We base this statement on the followingfacts. An individual who has either a mutant or polymorphic variant ofone of the genes for HUBC4 and/or APP exhibits AD. In thesecircumstances, the balance between functional HUBC4 and APP is impaired.We consider that the balance between the HUBC4 and APP gene products isnecessary to prevent disease.

For example, in a normal diploid individual the gene complement is asfollows:

    ______________________________________            HUBC4 APP            HUBC4 APP    ______________________________________

Whereas in an individual suffering from AD, any one or more of the abovegenes is defective resulting either in insufficient functional HUBC4gene product and/or deposition of APP gene product. Similarly, in asufferer of Down's syndrome this balance is impaired, but for differentreasons. A sufferer of Down's syndrome has an extra chromosome 21 andthe individual's gene complement is as follows:

    ______________________________________            HUBC4 APP            HUBC4 APP                  APP    ______________________________________

This imbalance brings about the pathological features, characteristic ofAD, in sufferers of Down's syndrome. It follows therefore that one wayof redressing the balance is to provide a Down's syndrome sufferer, viathe technique of the invention, with additional HUBC4 gene or geneproduct.

It is therefore the object of the present invention to provide a methodwhich enables a determination of a predisposition for and diagnosis ofdegenerative diseases such as AD; also products and processes fortreating and obtaining treatments for a degenerative disease such as ADand Down's syndrome; and also a method which enables a determination ofa predeposition for cancers, particularly, but not exclusively papillomavirus induced cancers, and also products and processes for treating andobtaining treatments for such cancers.

According to a first aspect of the invention there is therefore providedgenetic material having, including or derived from, substantially thegenetic sequence structure shown in FIG. 1, or part thereof, or afunctionally equivalent or functionally related or associated nucleotidesequence.

In an alternative embodiment of the invention the genetic sequencestructure comprises a nucleotide substitution at nucleotide 320 andideally a thymine to cytosine base substitution.

In yet a further alternative embodiment of the invention said geneticsequence structure comprises a nucleotide substitution at nucleotide 605and ideally an adenine to guanine substitution.

We consider that the said genetic sequence structure without any of theaforementioned substitutions represents the genetic sequence structureof the ubiquitin conjugating gene HUBC4a. Tie genetic sequence structureincluding the said substitutions represents the coding sequencestructure of the ubiquitin conjugating gene HUBC4b.

In yet further preferred embodiments of the invention said geneticsequence structure may include any one or more of the variations shownin FIG. 5.

According to a second aspect of the invention there is provided aprotein. ideally a ubiquitin conjugating enzyme, having substantiallythe amino acid sequence structure shown in FIG. 1, or part thereof, or afunctionally equivalent or functionally related or associated amino acidsequence structure.

In an alternative embodiment of the invention said amino acid sequencestructure comprises an amino acid substitution at position 23 andideally a cysteine to arginine substitution.

In yet a further alternative embodiment of the invention said amino acidsequence structure comprises a substitution at position 118 and ideallya lysine to glutamic acid substitution.

We consider that the amino acid sequence structure without any of theaforementioned substitutions represents the sequence structure of theprotein encoded by the gene HUBC4a and that the amino acid sequencestructure including the aforementioned substitutions represents theamino acid sequence structure encoded by the gene HUBC4b.

In yet alternative embodiments of the invention said amino acid sequencestructure may include any one or more of the variations shown in FIG.15.

For the avoidance of doubt, DNA sequences of the invention includesequences useful in securing expression in prokaryotic or eukaryotichost cells of a polypeptide as hereinbefore defined. DNA sequences ofthe invention are specifically seen to compromise:

a) a DNA sequence set forth in FIG. 1 and fragments or variants thereof,and in particular the fragments which code for the polypeptide of FIG.1, or its complimentary strand;

b) a DNA sequence which hybridises to the DNA sequence set forth in FIG.1 or to fragments or variants thereof; which sequence by implicationshows at least 30% and preferably 50°% homology with said FIG. 1sequence or fragments; such a sequence includes but is not restricted tothe primers hereindescribed.

c) a DNA sequence which, but for the degeneracy of the genetic code,would hybridise to the DNA sequence set forth in FIG. 1 or to fragmentsor variants thereof and in particular to the fragments or variants whichcode for the polypeptide of FIG. 1.

Specifically comprehended in part b) are genomic DNA sequence encodingallelic variant forms of the polypeptide of FIG. 1. Specificallycomprehended in part c) are manufactured DNA sequences. Manufacturedsequences may readily be manufactured according to the methods of, forexample, Edge et al, Nature, 292, 756-762 (1981).

The degeneracy of the genetic code allows substantial freedom in thechoice of codons which can be used to construct a gene for theappropriate polypeptide of the present invention. Codons are normallyselected which are preferred by the host.

Polynucleotide probes may be constructed which are capable ofhybridisation to any portion of the aforementioned DNA sequence or of acorresponding RNA or cDNA sequence. It will be appreciated that thenucleotide probe will comprise a nucleotide sequence capable ofhybridisation to a sufficient length of the sequence to be determined toensure that the probe unambiguously detects the sequence of interest. Ingeneral, the probe will be capable of hybridising to at least eightconsecutive nucleotides of the sequence to be determined.

According to a third aspect of the invention there is provided apolynucleotide which comprises a nucleotide sequence capable ofhybridising, to a DNA sequence as hereinbefore defined or a fragmentthereof, or a corresponding RNA sequence, said probe optionally having alabelled or marker component. Preferably said probe comprises at leastone probe as illustrated in FIG. 13 or a substantially similar probehaving deletions or additions which do not prevent the probe fromhybridising to said DNA sequence.

According to a fourth aspect of the invention there is provided at leastone antibody, monoclonal or otherwise, raised against a whole or a partof the DNA sequence structure or amino acid sequence structure shown inFIG. 1.

In a preferred embodiment of the invention said antibodies are raisedagainst either a highly conserved region of the DNA sequence structureencoding for amino acids 60-90 or more preferably 72-88, shown in FIG.1, or alternatively, against amino acids 60-90 or more preferably 72-88,shown in FIG. 1.

Ideally said antibodies are raised against the C terminal amino acidsand more preferably amino acids 137 to 154.

Such antibodies or probes may be used to detect the presence or indicatethe absence of a polypeptide as hereinbefore defined or correspondingDNA or RNA as appropriate and hence the presence or absence of materialpossessing ubiquitin conjugating enzyme activity. Thus the probes orantibodies may be used to indicate whether an altered ubiquitinconjugating enzyme mediated condition may be at least partly caused byan absence of ubiquitin conjugating enzyme HUBC4 activity.

According to a fifth aspect of the invention there is provided adelivery means including a whole or a part of the DNA sequence structureand/or the amino acid sequence structure shown in FIGS. 1, 2a or 14.

According to a sixth aspect of the invention there is provided geneticmaterial having, including or derived from substantially the mousegenetic sequence structure shown in FIG. 3, or part thereof, or afunctionally equivalent or functionally associated nucleotide sequence.

According to a seventh aspect of the invention there is provided aprotein having substantially the amino acid sequence stricture shown inFIG. 4, or part thereof, or a sequence structure which encodes for aprotein functionally identical or similar to the protein shown in FIG.4.

According to a eighth aspect of the invention there is provided at leastone antibody, monoclonal or otherwise, raised against a whole or a partof the DNA sequence structure, or amino acid sequence stricture, shownin FIGS. 3 and 4 respectively.

According to an ninth aspect of the invention there is provided adelivery means including a whole or a part of the DNA sequence structureand/or amino acid sequence structure shown in FIGS. 3 and 4respectively.

In a preferred embodiment of the invention the aforementioned deliverymeans comprises a vector, ideally a replicative vector. Alternatively,in another embodiment of the invention the delivery means comprises aliposome. Alternatively, said delivery means is a plasmid.

In a preferred embodiment of the invention said delivery means is aretroviral vector which enables specific delivery of the DNA sequencestructure to tissues for the purpose of gene therapy.

In yet a further preferred embodiment of the invention the deliverymeans is a adenoviral vector.

In yet a further preferred embodiment of the invention the deliverymeans is a Herpes virus vector.

It will also be evident to those skilled in the art that monoclonal orpolyclonal antibodies raised using standard techniques either to thewhole protein encoding any one of the HUBC4 proteins herein described orto epitopes or fragments thereof, to truncated variants thereof, tosplicing variants thereof and to protein variants displaying mutationsor amino acid changes may be produced. In addition, antibody fragments,which preferably contain the binding region of the antibody, as well aschimeric antibodies, for example as described in GB 2188638 may also beused. In the instance where fragments are used such fragments may beFab-type fragments, ie fragments lacking an Fc portion such as Fab, Fab1 and F(ab1)2 fragments or so called "half molecule" fragments obtainedby reductive cleavage of the disulphide bonds connecting the heavy chaincomponents in an intact antibody.

Further, secondary antibodies may also be raised to the saidaforementioned antibodies.

Antibodies such as the above will have utility in establishing thediagnosis of AD in post-mortem samples and may facilitate analysis ofneurological biopsies or other fluid/tissue samples. Moreover, given theknown implication of ubiquitination in a variety of other humandiseases, these antibodies may prove valuable in the diagnosis ofParkinson's Disease, Alcoholic Liver Disease, Pick's Disease andCerebellar Astrocytomas.

Antibodies such as the above will also have utility in blocking theactivity of at least one selected ubiquitin conjugating enzyme and sosafeguard against, at least, the deleterious ubiquitination of p53particularly in, but not limited to, HPV infected cells and cancers.

Ideally, the afore referred to delivery means comprise agents, such asantibodies that block the activity of HUBC4, and particularly, but notexclusively, HUBC4b and/or HUBC4b complexes.

Moreover, said antibodies may prove valuable in monitoring the level ofHUBC4 expression in individuals receiving gene replacement therapyaccording to the invention to treat any of the diseases describedherein. Selective monitoring of the different sorts of HUBC4 forming theHUBC4 classes herein described can be undertaken by manufacturingantibodies which recognise differences in the proteins encoded by theHUBC4 genes.

An alternative method of blocking or reducing the synthesis of HUBC4proteins comprises use of antisense sequences relating to the whole or apart of the DNA sequences shown in FIGS. 1, 2 or 14.

According to a tenth aspect of the invention there is provided anon-human transgenic animal whose genetic material includes at least onecopy of the DNA sequence structure shown in FIGS. 1, 2 or 14 and/or atleast one mutation or polymorphic variant of the DNA sequence structureshown in FIGS. 1 or 2 or 14 or alternatively, a non-human transgenicanimal whose genetic material does not include a part of the DNAsequence structure shown in FIG. 3 such that the protein encoded by saidsequence structure is either non-functional or not expressed.

Alternatively there is provided a non-human transgenic animal whose germcells and somatic cells contain at least one copy of the DNA sequencestructure shown in FIGS. 1, 2 or 14 and/or at least one mutation orpolymorphic variant of the DNA sequence structure shown in FIGS. 1 or 2or 14; and/or a recombinant activated mutant form of the DNA sequencestructure shown in FIGS. 1 or 2 or 14, provided in said animal at anembryonic stage, and/or an absence of the DNA sequence structure shownin FIG. 3 such that the protein encoded by the sequence structure iseither non-functional or not expressed, and its offspring possessing thesame gene sequence.

Preferably said transgenic comprises multiple copies of said sequencestructure and/or attachment of said sequence structure to a promoter,inducible or otherwise, that provides for enhanced expression of saidsequence structure. More preferably said transgenic comprises a reportergene so that temporal and/or spatial expression of said sequence can bemonitored. Ideally said reporter gene encodes a visual marker.

In a preferred embodiment of the invention said non-human transgenicanimals are provided using the YACs hereinafter described.

In a preferred embodiment of the invention said animal is a rodent suchas a mouse or a rat.

According to an eleventh aspect of the invention there is providedoffspring of said transgenic animal.

It will be evident to those skilled in the art that the availability oftransgenic animals will allow for animal models that developdegenerative diseases such as AD homologous to the condition in man andalso animal models that are susceptible to cancers and in particularpapilloma virus induced cancers. These models can be used to identifytreatments that prevent the onset or continuance of degenerativediseases such as AD in transgenic animals and which are therefore likelyto have therapeutic benefit in human AD and also treatments which candelay, prevent or treat cancers such as papilloma virus induced cancers.

According to a twelfth aspect of the invention there is provided amethod of diagnosing a predisposition for, or the existence of, adegenerative disease such as Alzheimer's Disease comprising a comparisonof test DNA sequence structure from an individual to be tested with thesequence structure shown in FIGS. 1, 2 or 14, or parts thereof.

In a preferred method of the invention the method comprises a comparisonof test DNA sequence structure from human chromosome 14, and ideallylocation 14q24.3 of human chromosome 14, from an individual to be testedwith the DNA sequence structure, or part thereof, shown in FIG. 1 and/orincluding/excluding, respectively, the above mentioned substitutions.

According to a thirteenth aspect of the invention there is provided amethod for diagnosing a predeposition for, or susceptibility to,cancers, and in particular papilloma virus induced cancers comprising acomparison of test DNA sequence structure from an individual to betested with the DNA sequence structure shown in FIGS. 1, 2 or 14, orparts thereof.

In a preferred method the diagnosis involves a comparison of test DNAsequence structure from human chromosome 22 and ideally location 22 q ofhuman chromosome 22, from an individual to be tested with the DNAsequence structure, or part thereof, shown in FIG. 1 and/orincluding/excluding, respectively, the above mentioned substitutions.

Preferably a type of HUBC4 cDNA sequence and the corresponding type ofHUBC4 genomic sequence are used in concert for the identification ofvariations in a sample from an individual to establish whether thatsample contains mutant forms of the different HUBC4 sequences. In apreferred aspect of the invention the existence of a mutation orpolymorphic variation is determined by single strand conformationalpolymorphism analysis. In a more preferred aspect of the presentinvention, mutation or polymorphic variation is detected by any methodknown in the art for the identification of mutation or polymorphicvariation in DNA sequences including direct DNA sequencing (7 and 8).

In a further aspect of the present invention, diagnosis of the presenceof a particular mutation or mutations in the HUBC4 genes mayconveniently be affected by PCR based techniques such as the ARMS method(6) for assessing the presence of a point mutation. Such point mutationsare identified by the approaches described above.

It will also be evident to those skilled in the art that the method ofdiagnosis essentially involves detection of the presence or absence of amutant HUBC4 allele in a sample from an individual (an allele is definedas a variant of a genetic locus and is inherited according toconventional principles of genetic segregation).

Variation of a genetic locus may be identified using DNA sequencevariation or any other method known in the art for detecting variationat a genetic locus including the analysis of microsatellite regions orminisatellite regions or restriction fragment length polymorphism(RFLP). The method of the invention may also be performed using anyexpressed gene product such as the HUBC4 protein or RNA.

It is also within the scope of the invention to express the proteinwhose sequence structure is shown in FIGS. 1, or 14 in eitherprokaryotic or eukaryotic heterologous expression systems.

According to a fourteenth aspect of the invention there is provided arecombinant DNA sequence identical or substantially similar to thesequence shown in FIGS. 1 or 3, or any part thereof. Ideally saidsequence shown in FIG. 1 includes the aforementioned substitutions.

According to a fifteenth aspect of the invention there is provided anexpression and ideally export system, such as for example a yeast,bacterial or mammalian cell, which is capable of expressing and ideallyalso exporting the gene and protein products respectively of theinvention.

In a preferred embodiment of the invention said system includes multiplecopies of the said gene and/or an enhanced promoter whereby expressionof the said gene product is enhanced. It will be apparent to thoseskilled in the art that such a system could be used for therapies basedon surgical transplantation of cells into critical loci preferablyneuronal loci.

It will be appreciated that where the desired product is not passed outof the host cell at a commercially useful rate, the host cell may becultured and harvested as the intact cell and the desired polypeptiderecovered by subsequently extracting the cells, for example afterseparation from the medium containing nutrients necessary for growth ofthe host cell. Where the product is passed out of the host cell into thesurrounding culture solution, then the polypeptide may be recovered byextraction in the normal way.

According to a sixteenth feature of the present invention there istherefore provided a transformed host capable of expressing apolypeptide as hereinbefore defined, the host comprising a replicableplasmid-derived expression vehicle, said vehicle comprising geneticmaterial coding for the said polypeptide.

According to a seventh aspect of the invention there is providedrecombinant host cells transformed with the expression system of theinvention. Said recombinant host may be a prokaryotic or a eukaryotichost such as embryonic stem cells or mammalian cells.

According to an eighteen aspect of the invention there is a provided amethod of producing a human ubiquitin conjugating enzyme of theinvention which method comprises culturing a recombinant host celltransformed with the one of the HUBC4 expression systems of theinvention.

According to an nineteenth aspect of the invention there is provided theDNA sequence structure and the protein sequence structure shown in FIGS.1, 2 or 14, or any part thereof, for the treatment of degenerativediseases such as Alzheimer's Disease, or Down's syndrome.

In a preferred embodiment of the invention said amino acid sequencestructure shown in FIGS. 1, 2 or 14 is provided in a pharmaceuticalcomposition in association with a pharmaceutical excipient or carrier.

According to a twentieth aspect of the invention there is provided amethod of treating a degenerative disease such as Alzheimer's Diseaseusing the DNA and/or protein sequence structures, or any parts thereof,shown in FIGS. 1, 2 or 14.

According to a twenty-first aspect of the invention there is provided amethod of treating Down's syndrome using the DNA and/or protein sequencestructures, or any parts thereof, shown in FIGS. 1, 2 or 14.

The polypeptides, and fragments thereof, of the present invention areobtainable in a biologically pure and homogenous form. The presentinvention also provides a polypeptide as herein defined unaccompanied byassociated native glycosylation. In addition to polypeptides ashereinbefore defined, the present invention also embraces other productswhich possess enzymic activities as human ubiquitin conjugating enzymessuch as polypeptide analogues or other members or the family of humanubiquitin conjugating enzymes which may be capable of substituting forHUBC4 or working with, the polypeptides of FIGS. 1, 2 or 14, in vivo orin vitro.

These polypeptide analogues include polypeptides which differ from thathereinbefore defined in terms of the identity or location of one or moreamino acid residues. For example, such analogues may containsubstitutions or terminal or intermediate additions or deletions of suchresidues. Such analogues would share ubiquitin conjugating enzymeactivity. As examples, projected products of the invention include thosewhich are more stable to hydrolysis (and therefore may have morepronounced or longer lasting effects than naturally occurring); or whichhave one or more cysteine residues deleted or replaced by, for example,alanine or serine residues and are potentially more easily isolated inactive form from microbial systems.

According to a twenty-second aspect of the invention there is provided aprotein or polypeptide having the sequence stricture shown in FIGS. 1,2, 14 or 3, or parts thereof, or a homologue or analogous thereof.

The polypeptides of the present invention may conveniently be preparedby genetic engineering techniques. Analogues of the present inventionmay be prepared by expression of genes coding for such analogies. Suchgenes may readily be obtained by modifications of cDNA and genomic genesby well-known site directed mutagenesis techniques.

According to a yet further aspect of the present invention there areprovided Yeast Artificial Chromosome (YAC) clones and/or cosmid cloneswhich contain the class of HUBC4 genes herein described plus theirgenetic control elements including promoters and enhancers.

In a further aspect of the present invention highly polymorphicmicrosatellite repeats are isolated from within these YAC and/or cosmidclones for use in diagnosis of degenerative diseases such as AD infamilies.

It is of note that AD does not naturally occur in rodents and indeed inour own investigations we have shown that UBC4 expression occurs in awide variety of rodent tissues. In contrast, in man, in particular in anormal individual, we have shown that there is a marked reduction inHUBC4 expression in brain tissue compared to other tissues suggestingthat an aberration in HUBC4 expression is likely to be of mostsignificance in brain tissue. This data correlates well with theneuronal lesions characteristic of the disease.

The invention will now be described, by way of example only, withreference to the accompanying Figures wherein;

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA sequence structure and the corresponding amino acidsequence structure of the HUBC4a genie found on human chromosome 14;

FIG. 2 shows the nucleotide and deduced amino acid sequence of HUBC4bcDNA. There is shown the nucleotide sequence and predicted amino acidsequence of the 3' region of the HUBC4b genie. The nucleotide sequenceof the 3' end of an intron present within the HUBC4 is detailed in lowercase letters. The 3' intron/exon junction splice site commutative intronbranch point sequence are underlined;

FIG. 3 shows the partial cDNA sequence of mouse UBC4; and FIG. 4 showsthe corresponding amino acid sequence structure of mouse UBC4;

FIGS. 5A-5C shows a fluorescent in situ hybridisation (FISH)demonstrating (a) chromosome localisation of HUBC4a to human chromosome14q24.3 using the YAC LMM-YAC1. (b) Localisation of HUBC4c to humanchromosome 12q using the YAC LMM-YAC4. (c) Localisation of HUBC4b tohuman chromosome 22q using the cosmid LMM-COS1;

FIGS. 6A-6D shows chromosomal location of HUBC4a, HUBC4b, HUBC4c, HUBC4dand HUBC4e using the NIGMS iuman/rodent somatic cell hybrid mappingpanel #2 version 2.

(a) Localisation of HUBC4a to human chromosome 14 with PCR primers

5'd=(GAAGCCAGCAACCAAACCCGA) and

5'd=(CACAAGCGAACGCCAGGC)

(b) Localisation of HUBC4b to human chromosome 22 using PCR primers

5'd=(TAGGAGGCAGCTTTGGCTT) and

5'd=(CACAAGCGAACGCCAGGC)

(c) Coamplification of HUBC4a and HUBC4b using PCR primers

5'd=(CCAGCCTGAGCACCCGCTT) and

5'd=(CACAAGCGAACGCCAGCC)

(d) Localisation of HUBC4a, HUBC4c, HUBC4d, and HUBC4e to humanchromosome 14, 12, 19, and 13 respectively using PCR primers

5'd=(GAAGCCAGCAACCAAAACCGA) and

5'd=(GGTGTCTGAATGCACTGCA)

The human chromosomal DNA used as a PCR substrate is indicated aboveeach line (1-22, X, and Y). `H` total human DNA positive control. `Mo`mouse DNA negative control. `Ha` Hamster DNA negative control. `C` noDNA control. `M` DNA markers (Φx 174 DNA/Hae Ill digested).

FIGS. 7A-7B shows a northern blot of mouse and human mRNA hybridisedwith an HUBC4a probe illustrating, tissue expression distribution;

FIG. 8 shows a pulsed field gel electrophoresis of the four YAC clonesLMM-YAC1, 2, 3 and 4. The gel was Southern blotted and probed with anHUBC4b derived fragment as described hereinafter;

FIGS. 9A-9C shows a comparison of the hydrophobicity profiles of yeastUBC4 and human HUBC4a or HUBC4b to illustrate their similarity;

FIG. 10 shows a comparison of the primary amino acid sequence of HUBC4bwith the primary amino acid sequences of a number of yeast, human anddrosophilla ubiquitin conjugating enzymes and specifically UbcH2, UbcH5,UBC4, UbcD1, BEN and UbcD-2. The sequence data illustrates thesimilarity between the sequences and the predictable functionalsimilarity between the corresponding proteins;

FIG. 11 shows alignment of predicted amino acid sequences encoded by theidentified human (HUBC4a, HUBC4b, HUBC4c) and murine (mUBC4) genes. Thefull sequence of the protein encoded by HUBC4a is presented as thereference sequence. Identical residues encoded by HUBC4b, HUBC4c, andMUBC4, with respect to the reference sequence, are indicated with anasterisk (*).`-` represents shifting of the amino acid sequence tomaintain alignment. `.` represents unknown amino acid composition;

FIG. 12 shows alignment of nucleotide sequences of the identified humanHUBC4a gene, HUBC4b cDNA, HUBC4c gene, and the murine HUBC4 gene. Thenucleotide sequence of HUBC4a is detailed as the reference sequence.Identical nucleotides encoded by HUBC4b, HUBC4c, and mUBC4, with respectto the reference sequence are indicated with an asterisk (*). `-`represents shifting of the nucleotide sequence maintain the alignment.`.` represents unknown nucleotides;

FIG. 13 shows primers used to amplify human and murine UBC4 genes.Primers (1), (3), (5), (7), (8), (9), and (11) were 5'-3' primers, and(2), (4), (6),(10) and (12) were 3'-5' primers with respect to thesequences detailed in FIGS. 1-3. Primer combinations. Primers (5), (6),(7), (8), (9) and (10) were used to specifically amplify human HUBC4genes, or regions thereof, even in the presence of mUBC4. The PCR wasperformed using a preliminary denaturation step of 95° C. for 5 min,followed by cooling to 90° C., prior to adding Taq polymerase enzyme.Thirty-five cycles of the PCR were then performed at 62° C. for 15 sec,72° C. for 20 sec, and 94° C. for 15 sec.

FIG. 14 shows nucleotide and predicted amino acid sequence of thecharateristed region of the human gene encoding HUBC4c.

FIG. 15 shows variations in the HUBC4 genes and predicted amino acidsequences with respect to HUBC4a.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is concerned with the novel nucleotide sequencesassociated with the diseases described herein and presented in theFigures, especially FIG. 2. The gene of FIG. 2 encodes the novel proteinHUBC4b. The corresponding cDNA clone is entitled LMM-cDNA1 and wasdeposited with the National Collection of Industrial and MarineBacteria, 23 St Macher Drive, Aberdeen, AB2 1RY, Scotland on 10.5.94.Its deposition number is NC IMB 40626. The invention is also concernedwith a novel mouse protein mUBC4. The corresponding cDNA clone isentitled LMM-cDNA2 and was deposited with the National Collection ofIndustrial and Marine Bacteria, 23 St Macher Drive, Aberdeen, AB2 1RY,Scotland on 10.5.94. Its deposition number is NC IMB 40637. Theinvention also concerns the whole genes described herein encoding thehuman cDNAs and their transcriptional control elements. Genes HUBC4a andHUBC4c are contained within the Yeast Artificial Chromosomes LMM-YAC1(HUBC4a), LMM-YAC2, LMM-YAC3 and LMM-YAC4 (HUBC4c). These four YACs werealso deposited with the National Collection of Industrial and MarineBacteria, 23 St Macher Drive, Aberdeen, AB2 1RY, Scotland on 31.3.94.Their deposition numbers are respectively NC IMB 40627, NC IMB 40628, NCIMB 40629 and NC IMB 40630. The cosmids LMM-COS 1, LMM-COS2, LMM-COS3were also desposited as above on 28.3.95 and their deposition numbersare 40711, 40712, 40713. They contain HUBC4b.

Isolation and Characterisation of HUBC4a, HUBC4b, HUBC4c, HUBC4d andHUBC4e (see FIGS. 1, 2 and 14)

The method will be described with particular reference to HUBC4b. HUBC4bclones were isolated during a differential hybridisation screen ofnormal oral palatal mucosa and octontogenic keratocyst cDNA librariesthat had been prepared in lambda GEM2 bacteriophage vector. cDNA insertsof picked plaques were amplified by PCR using vector specific primers(T7 promoter primer and SP6 promoter primers)--30 cycles of 1 min 94°C., 2 min 50° C., 3 min 72° C.). These inserts were then purified byagarose gel electrophoresis.

Purified inserts were sequenced using the double stranded ds DNA cyclesequencing system (Life Technologies, Paisley, Scotland). In addition,inserts were radio-labelled using a random priming labelling kitmanufactured by Boehriger Mannheim Ltd and ∝-³² P!dCTP, and then used toscreen Lambda ZAP cDNA library prepared from RNA extracted from normaloral palatal mucosa. Plaques giving a positive signal after highstringency washing were isolated, phagemids excised and sequenced usingSequenase Version 2.0 sequencing test (USB). cDNA sequence and deducedprotein sequence is shown in FIG. 2a.

The method for isolation of the cosmids which encode the HUBC4b gene isas follows:

500,000 colony forming units (cfu) of a human cosmid genomic DNAlibrary, prepared from human placental DNA in the vector pWE15(Clontech, Cambridge Bioscience, Cambridge, UK), was screened with ³²P!-labelled HUBC4b cDNA probe. Positive clones were selected, plated ata lower density, and rescreened. A third round of screening wasperformed, if necessary, to isolate individual clones. Cloneshybridising with the probe were cultured, and DNA was prepared accordingto standard methods. Cosmid DNA was restriction digested and subclonedinto the pBluescript II vector (Stratagene Ltd, Cambridge, UK) forsequencing

Isolisation and Characterisation of Mouse UBC4 (see FIGS. 3 and 4)

Human PCR primer pairs (1) and (4) as described below were used toamplify mouse mUBC4 directly from mouse genomic DNA. This DNA was thensequenced using a ds DNA cycle sequencing kit as described above. cDNAsequence and deduced protein sequence is indicated in FIG. 3 and FIG. 4.A high degree of homology was noted between the mouse and humansequences (compare FIGS. 1 and 2 with FIGS. 3 and 4).

Chromosome Localisation of HUBC4 ie HUBC4a, HUBC4b, HUBC4c, HUBC4d, andHUBC4e (see FIGS. 5, 6 and 13)

PCR primers were designed for the amplification of human HUBC4 and mousemUBC4 genes.

1. (5'-dGGCAGGTCTGTCTGCCAG)

2. (5'-dGGTCTCTGCTCACACTTGCTG)

3. (5'-dCAGCAAGTGTGAGCAGAGACC)

4. (5-dCTTTACAGGTTACCTAGACCAC)

5. (5-dGAAGCCAGCAACCAAAACCGA) and

6. (5-dCACAAGCGAACGCCAGGC).

Primers (1), (3) and (5) were 5-3' primers and (2), (4) and (6) were3-5' primers with respect to the cDNA sequence. Primers (5) and (6) wereused to specifically amplify human HUBC4a even in the presence of mousemUBC40. Standard PCR conditions used a hot start after an initial 7 minincubation at 96° C., then cooling to 80° C. prior to the addition ofpolymerase enzyme followed by a 40 cycle PCR reaction of 45 sec at 94°C., 30 sec at 55° C. and 2 min at 72° C. Amplified products wereanalysed by agarose gel electrophoresis. Taq Polymerase and/or bufferwas obtained from Promega Biotech. Deoxynucleotides were used at a finalconcentration of 200 μm and primers at 20 pmol/100 μl PCR reaction.

DNA extracted from NIGMS somatic cell hybrid panel no 2 (NationalInstitute of General Medical Services USA obtained from the Coriell CellRepository, 401 Haddon Avenue, Camden, N.J. 08103, USA) was used tochromosome localise HUBC4a. Primer pairs 5 and 6 were used for thispurpose to avoid amplifying mouse mUBC4 from the mouse DNA present insomatic cell hybrids (see FIG. 6). Only one band of the same size asthat of the human genomic DNA control was observed in the lanecorresponding to chromosome 14 (see FIG. 6).

Fluorescence in situ hybridisation of metaphase spreads prepared usinghuman chromosomes was preformed using biotinylated probes prepared byrandom priming of total HUBC4a YAC DNA from LMM-YAC1. A signal wasobtained at a banding position on the chromosome corresponding to14q24.3 (see FIG. 5).

Comparison of HUBC4a, HUBC4b and UBC4 (see FIGS. 9 and 10)

A comparison of HUBC4a and HUBC4b and UBC4 protein sequences is shown inFIG. 9. A 62% homology is found. A further comparison of thehydrophobicity profiles of human HUBC4b and yeast UBC4 protein sequencesalso demonstrated remarkably similar profiles (see FIG. 10). Forexample, the peaks and troughs at approximate amino acid positions 40,70, 85, 100, 120 coincide in both proteins.

Comparison of Human and Mouse Expression Distribution of HUBC4b andMUBC4 using Northern Blots of RNA Extracted from Various Mouse and Humantissues (see FIG. 7)

Clontech multiple tissue Northern blots of RNA extracted from mouse andhuman (Cat Nos 7762-1 and 7760-1 respectively) were carried out as permanufacturer's instructions using a human HUBC4b DNA probe (prepared asdescribed above). Bands corresponding to HUBC4b are indicated. Thenature of the upper bands is not known. HUBC4 RNA transcripts were foundin all tissues examined. Note the high levels of expression in mousebrain but the comparatively lower level in human brain (FIG. 7).

Isolation of Yeast Artificial Chromosomes Containing HUBC4 Sequences(see FIG. 8)

A Leeds University Molecular Medicine Unit YAC library prepared usinghuman genomic DNA in the vector PYAC4 and in S cerevisiae strain AB1380was configured for PCR screening in standard fashion. Primary DNA poolswere screened with PCR primer pairs 11 and 12 (FIG. 13). Four positiveswere identified. Secondary and tertiary DNA pools were then screened toidentify individual YAC's containing HUBC4 sequences. These YACs werethen tested by PCR with the other HUBC4 primer pairs 3 and 4 and 5 and6. Positive PCR signal patterns were obtained by analysis on agarose gelelectrophoresis. No positive signal was observed with a control YAC. Thefour positive HUBC4-YACs (LMM 1-4) as judged from PCR were thensubjected to pulse field gel electrophoresis. Southern blot analysis ofthese runs was performed by hybridising with a HUBC4b ³²P-radio-labelled DNA probe, prepared as described above. Positiveautoradiographic signals were achieved with all four YACs (see FIG. 8),LMM (1-4). Sizing of the human genomic DNA inserts in these separateYACs was performed by comparison of their positions of migration withthose of the normal S cerevisiae chromosomes on ethidium bromide stainedpulse field gels.

Preparation of Polyclonal Antisera

Peptide CKNAEEFTKKYGEKRPVD was conjugated to hemocyanin (keyhole limpet)using the heterobifunctional reagent MBS(m-maleimidobenzoyl-N-hydroxysuccinimide ester). Two rabbits (MAK-1 andMAK-2) were immunised with this conjugate. Rabbits were bled at regularintervals (3, 5, 7, 9 and 11 weeks) and antibody titre determined byELISA (final titre approximately 1:1000). Antisera were collected after11 weeks and antibodies affinity purified. The titres of the affinitypurified antibody solutions were both approximately 1:3000.

Production of HUBC4 Gene Products by Baculovirus Expression

Expression of large quantities of protein encoded by the HUBC4b cDNA,and the HUBC4a gene was achieved using a baculovirus system.

Construction of Transfer Vector HUBC4b cDNA was PCR amplified frompurified lambda GEM-2 bacteriophage cDNA clones using vector specificprimers (T7 promoter primer and SP6 promoter primer). The cDNA wasdigested from the flanking vector sequence by restriction digestionusing Xba I and Eco RI. The insert was gel purified and ligated into thesimilarly digested transfer vector pBacPAK9 (Clontech). The ligationmixture was transformed into competent E. coli DH5α cells, andrecombinants were sequenced to confirm the integrity of the cloned cDNA.

DNA encoding the HUBC4a open reading frame region was generated asdescribed in `production of vectors encoding antisense HUBC4 genes`. ThePCR product was blunt ended using T4 DNA polymerase, and it was thencloned into the Sma 1 site of pBacPAK9. Recombinants were selectedcontaining inserts in the correct orientation for transcription of theopen reading frame.

Preparation of Linearised Viral DNA

Bsu 361 linearised BacPAK6 DNA was obtained from Clontech, or producedfrom circular viral DNA by restriction digestion with Bsu 361essentially as described by Kitts and Possee (993). 2 μm of virus DNAwas digested overnight at 37° C. with Bsu 361 in restriction enzymebuffer, and then heat inactivated by incubation at 65° C. for 10 min.The DNA was then stored at 4° C.

Cotransfection of Viral DNA and Recombinant Transfer Vector

Cotransfection was performed according to the procedure of King andPossee (1992). 35 mm petri dishes were seeded with 1.5×10⁶ Spodopterafrugiperda cells and then incubated at 28° C. for 2 b. 2.5 μg ofrecombinant transfer vector DNA and 0.5 μg of linearised viral DNA weremixed in a sterile 5 ml glass bijou in a volume of 25 μl. 2.5 μl oflipofectin (diluted 2:1 with sterile water) (Gibco-BRL) was added to theDNA and the mixture incubated for 15 min at room temperature.Immediately prior to transfection, the cell monolayers were washed with2 ml of serum-free TC100 medium whereafter the monolayer was coveredwith 1.5 ml of medium to which the DNA/lipofectin mixture was added. Theplates were incubated at 28° C. for 8 h and the medium was then replacedwith TC100 medium supplemented with 10% (v/v) foetal calf serum. Thedishes were incubated for a further 2 days whereafter the supernatantwas harvested for virus purification.

Virus Purification

35 mm petri dishes were seeded with 1.5×10⁶ Spodoptera frugiperda cellsand then incubated at 28° C. for 2 h. Thne medium was removed, and 0.1ml of diluted virus suspension (10⁰, 10¹ , 10², 10⁻³, and 10⁻⁴dilutions) was gently pipetted into the centre of each dish. Virus wasallowed to adsorb for 1 h at room temperature, after which the inoculumwas removed and the cell layer covered with 2 ml of 37 overlay 1% (w/v)low gelling temperature agarose, 50% (v/v) TC100 medium, and 2.5% (v/v)foetal calf serum!. After the overlay had solidified, 1 ml of TC100/2.5%foetal calf serum was added to supplement the medium, and the plateswere incubated for 3 days at 28° C. in a humidified atmosphere.Recombinant virus plaques were identified by staining with X-gal, in thepresence of neutral red. Putative recombinant plaques were identified aswhite plaques. Positive plaques were picked into 0.5 ml of TC100/foetalcalf serum and retitrated. Two rounds of plaque purification wereperformed to generate pure stocks of recombinant virus.

Isolation of HUBC4 Gene Products

For the production of large amounts of expressed HUBC4 gene productsSpodoptera frugiperda cells were grown to a density of approximately 10⁶/ml in 21 spinner flasks containing 50 ml of TC100/2.5% fetal calfserum. Cells were infected at a multiplicity of infection of 3-5, andthen incubated at 28° C. for 3 days. To recover the protein, cells werepelleted from the medium by centrifugation at 5000 g for 10 min. Thecells were then washed in phosphate buffered saline and then lysed bysonication in the presence of TENT buffer 100 mM Tris-Cl, pH7.5, 0.1MNaCl, 1% (v/v) Triton X-100, 1 mM EDTA! containing 1 mM NEM and 1 mMPMSF. The released protein was precipitated Using ammonium sulphate thenpurified by FPLC.

REFERENCES

King, L. A. and Possee, R. D. (1992). "The bacuolvirus expressionsystem, a laboratory guide", pp 1-229. Chapman and Hall, London.

Kitts, P. A. and Possess, R. D. (1993). A method for producingrecombinant virus expression vectors at high frequency. Biotechniques14, 810-817.

Production of Vectors Enconding Antisense HUBC4 genes

Construction of HUBC4 Anti-Sense RNA Expression Vectors

HUBC4b cDNA was PCR amplified from purified lambda GEM2 bacteriophageHUBC4b cDNA clones using vector specific primers (T7 promoter primer andSP6 promoter primer). The cDNA was digested from the flanking vectorsequence by restriction digestion with Xba I and Eco RI. The insert wascloned into Xba I IEco RI digested prokaryotic/eukaryotic expressionvector pBK-CMV (Stratagene). Plasmids were transformed into competent E.coli XLI-Blue MRF, selecting colonies containing inserts for sequenceanalysis to confirm correct insert orientation and sequence integrity.Transcription from the CMV immediate early promoter of the anti-senseHUBC4b construct (pLMM-AS1) results in transcription of the anti-sensestrand of the HUBC4b cDNA.

For HUBC4a anti-sense expression vector construction, the gene was firstPCR amplified from human genomic DNA using oligodeoxynucleotide primers(5'-dATGGCGGCCAGCAGGAGGCTG 3' and 5'd-CTTTACAGGTTACCTAGACCAC 3'). ThePCR product was blunt ended using T4 DNA polymerase, and ligated intothe Sma I site of the pBK-CMV vector. Ligation products were transfromedinto E. coli XLI-Blue MRF. Colonies containing cloned insets weresequenced, selecting those containing the insert in the anti-senseorientation (designated pLMM-AS2).

Cell Culture

HeLa cells were maintained in DMEM supplemented with 10% fetal calfserum, 10 μg/ml steptomycin, and 50 μg/ml penicillin. To constructanti-sense cell lines, cells were transfected with pLMM-AS1 or pLMM-AS2using the cationic-liposome DOTAP as detailed in `liposome-mediateddelivery of nucleic acids and proteins to eukaryotic cells`. Selectionfor maintenance of the vector was achieved by inclusion of the drug G418in the cell culture medium.

Liposome Mediated Delivery of Nucleic Acids and Proteins to EukaryoticCells

Transfection of cells with nucleic acids containing sequence encodinggene(s), cDNA(s), oligonticleotides containing sequence complementary toHUBC4/mUBC4, or parts thereof, or of proteins encoded by HUBC4/mUBC4genes, or parts thereof, was achieved using DOTAP, N-1-2,3-Diolcoyloxy)propyl!-N,N,N-trimethyl ammonium methylsulphate(Boehringer Mannheim), a cationic-liposome transfection reagent.

Method for Transfection of Cells

Cell Preparation

1-3×10⁵ aliquots of adherent tissue culture cells were passaged into 60mm diameter tissue culture dishes containing 5-6 ml of tissue culturemedium the day prior to transfection.

Preparation of Nucleic Acid/Liposome Complex

5 μg of DNA was diluted to 0.1 μg/ml in HEPES buffer 20 mM HEPES(cellculture grade), pH7.4! in a sterile reaction tube. In a second tube, 30μl DOTAP was diluted to 100 μl in HEPES buffer. The 50 μl nucleic acidsolution was mixed with the DOTAP suspension, gently mixed by pippeting,and then incubated for 15 min at room temperature.

Cell Transfection

Immediately prior to transfection, the DOTAP/nucleic acid mixture wasmixed with 5-6 ml of sterile tissue culture medium. The medium coveringthe cells was poured off, and the DOTAP/nucleic acid containing mediumwas added to the cells. The cells were incubated at 37° C. for 6 hourswhereafter the medium was replaced with fresh culture medium.

Expression of HUBC4 in Prokaryotic Cells

Blunted ended HUBC4 insert (10 ng) was generated by both RT-PCR (reversetranscribed polyerase chain reaction) and also PCR of genomic DNA andsub-cloned into the Sma 1 restriction endonuclease site of pGEX-3X (100ng). This DNA construct was then used to transform competent E. coliDH5α cells (100 μl). Transformed cells were selected by growth on LBagar plates containing 50 μg/ml ampicillin.

Plasmid DNA was isolated from an overnight culture of selected coloniesand sequenced to establish the presence of insert in the correct readingframe for expression of glutathione S-transferase fusion proteinproduct.

A 1:100 dilution of overnight culture of transformed cells containinginsert was grown to optical density of 0.5 (at wavelength 600 nm).Isopropyl-β-D-thiogalacloside was then added to a concentration of 1 mMand the cells grown for an extra 5 hr.

Cell extracts were prepared as follows. Broth was centrifuged (2000 gfor 10 min) and cell pellet collected. These cells were then resuspendedin PBS (140 mM sodium chloride, 2.7 mM potassium chloride, 10 mMdisodium orthophosphate and 1.8 mM potassium dihydrogen phosphate pH7.3). Cells were sonicated and Triton X100 added to a finalconcentration of 1% and mixed for 30 min. Extract was then centrifuged.Supernatant was used for the affinity purification of fusion protein.

Supernatant was mixed with glutathione-Sepharose 4B equilibrated withPBS containing 1% Trion for 30 min. This suspension was then sedimentedby centrifugation at 500 g for 5 min. The Sepharose pellet was washedwith PBS three times. Fusion protein was then eluted by incubation ofthe Sepharose beads for 10 min at 20° C. with 50MM tris/HCI buffer, pH8.0, containing 10 mM glutathione. This elution procedure was repeated afurther two times.

Production of Transgenic Animals by Microinjection

DNA microinjection and transgenic animal production were carried outessentially as described in Chapter 2 of Transgenic animal technology; Alaboratory handbook. Edited by Carl A. Pinkert; Academic Press Inc.1994. Briefly, this involved:

1. Super ovulation of 6 week old female F1 hybrid mice (CBA/J×C57B1/6)by intraperitoneal injection of 5 units of pregnant mare's serumgonadotrophin (PMSG) followed 47 hours later by 5 units of humanchorionic gonadotrophin (HCG).

2. Super ovulated donor dams were caged with fertile male mice followinginjection with HCG. The following morning, females were checked forcopulatory plugs, the presence of which was taken as a sign of mating.

3. Mated females were sacrificed using a schedule 1 method and theoviducts removed. Eggs were released from the oviduct into hyaluronidaseM2 medium by tearing with forceps. After washing with M2 medium, eggswere incubated at 37° C., 5% CO₂ in M16 medium prior to DNAmicroinjection.

4. Plasmid DNA constructs fro microinjection were prepared usingstandard cloning techniques and purified to a high decree by isolatingsupercoiled DNA from caesium chloride gradients (performed twice).Plasmid constructs were linearised to remove vector sequences prior topurification using phenol/chloroform ethanol precipitation. YAC DNA wasprepared for injection according to the method of Schedl et al., 1993,Nature 362, 258-261.

5. DNA at a concentration of 2-4 ng/μl was injected into fertilisedoocytes using a specialised microscope (Zeiss) fitted withmicromanipulators (Narashige). Needle preparation for holding andinjection pipettes was performed using a Sutter model P-87 pipettepuller following the method described in Pinkern 1994.

6. Injected oocytes were transferred into the oviduct of pseudopregnantrecipient dams made pseudopregnant by mating with vasectomised males.Recipient dams were placed under general anaesthetic and a 5mm surgicalincision made above the ovarian fat pad. The fat pad along the ovary andoviduct were exteriorised and held in position with a serafin clip. Thebursa surrounding the ovary was torn with forceps to reveal theinfundibulum. Using a glass capillary needle injected oocytes (15-20)were blown into the infundibulum using mouth pipetting and with the aidof a stereo dissecting microscope. The reproductive tract was carefullyreplaced in the abdomen and the body wall and skin closed with sutures.

7. Recipient dams were allowed to recover and to give birth. Weanedoffspring were analysed for the presence of the transgene by Southernblotting or PCR analysis of genomic tail DNA.

Production or Transgenic Animals Using Homologous Recombination inEmbryonic Stem (ES) Cells

Introduction of mutations into HUBC4 and HUBC4 genes and generation ofnull mutations was achieved using homologous recombination in ES cells.Targeting vectors were constructed using standard cloning procedures. Areplacement type vector (Deng et al., 1993; Mol. Cell. Biol. 13(4),2134-2140) was used to substitute the coding region of the gene with theneo^(r) gene thus conferring neomycin resistance on targeted cells. Anegative selection marker, the herpes simplex virus thymidine kinase(HSV TK) was included at the 3' end of the targeting construct to selectagainst random integration events. To introduce mutations into the UBCcoding and flanking sequences insertion type vectors were also made(Deng et al. 1993, as above). In addition gene targeting and productionof conditional targeted mutants using the CreloxP recombination systemof bacteriophage P1 as described by Gu et al., 1993. Cells 73, 1155-1164was employed. The different types of targeting construct were introducedinto ES cells and used to produce transgenic mice by the same method asdescribed in Gene Targeting--A Practical Approach Edited by A. L. JoynerIRL Press Oxford 1993. Briefly, this involves:

1. Culture of ES cells (mouse strain 129 derived) on mitoticallyarrested neo^(r) feeder layers of primary mouse embryonic fibroblasts inDMEM+20% foetal calf serum at 37° C.

2. Transfection of targeting constructs into ES cells by electroporationin a Biorad gene pluser at 500 μF, 240 v for 6 msec. Allow cells torecover and seed petri dishes containing mitotically arrested feederlayers with the ES cells. After 24 hours, positive selection is startedby the addition of 350-400 μm/ml of G418 and negative selection witheither 2 μm gancycolvir or 0.2 μm FIAU. The medium is replaced daily andresistant colonies identified after 9-11 days.

3. Surviving ES cell colonies were screened individually for homologousrecombination at the targeted locus using PCR and confirmed usingSouthern blotting using genomic DNA from the ES cells.

4. Targeted ES cells clones were expanded and aliquots frozen prior toinjection into blastocysts. Cells were prepared for injection by washingtwice with PBS, followed by trypsinisation of the cells from the culturedish. The ES cells were dissociated in culture medium by repeatedpipetting and the cell suspension transferred to a culture dish andincubated at 37° C. for 1 hour to allow the ES cells to separate fromthe feeder layer. After removing the supernatant, ES cells were detachedfrom the culture dish by repeated pipetting with culture medium,pelleted by centrifucation and then resuspended in 100-500 μl culturemedium prior to blastocyst injection.

5. C57B 1/6 blastocysts were flushed from the uteri of mated females at3.5 days post coitum and incubated in M 16 medium at 37° until used forinjection.

6. Blastocysts were injected with ES cells using an inverted microscope(Zeiss) and specialised micromaniptilators (Narashige) designed forproduction of transgenic animal. 10-15 ES cells were injected into eachblastocyst and the injected blastocysts incubated in M16 medium for 1-2hours prior to implantation in pseudopregnant foster mothers.

7. Injected blastocysts were implanted into the uterus of 2.5 day postcoitum pseudopregnant females made by mating normal females withvasectomised males. Resulting male chimeric offspring (as determined bycoat colour) were mated with C57VL/6 females to test for germlinetransmission of the transgene. The offspring of germline chimeras wereused to establish a breeding colony and produce experimental animals ondifferent genetic backgrounds.

Diagnosis of Alzheimer's Disease

Using the DNA and protein sequence structure disclosed in FIGS. 1, 2 or14 polynucleotide probes (see FIG. 13 for examples) for diagnosticpurposes may, if desired, be constructed which are capable ofhybridisation to any portion of the DNA protein or RNA sequenceregardless of whether the portion is capable of translation into apolypeptide or not. Moreover, if desired the protein HUBC4 in the formof an RNA sequence may be transcribed into a corresponding cDNA sequenceusing, for example, reverse transcriptase and the protein determined bythe use of a polynucleotide probe capable of hybridising to any portionof the cDNA sequence. It will be appreciated that the polynucleotideprobe will comprise a nucleotide sequence capable of hybridisation to asufficient length of the sequence to be determined, to ensure that theprobe will be capable of hybridisation to at least eight consecutivenucleotides of the sequence to be determined, preferably to at least 10consecutive nucleotides, more preferably to at least 12 consecutivenucleotides and especially to at least 14 consecutive nucleotides. Thepolynucleotide process of the present invention may be labelled ormarked according to techniques known in the art, for example, .sup. 32!P radio-labelled in any conventional way, or alternativelyradio-labelled by other means well known in the hybridisation art, forexample, to give .sup. 35! S radio-labelled probes. The probes may ifdesired carry fluorescent markers. They may alternatively be labelledwith Biotin or similar species by the method of D C Ward et al asdescribed in proceedings of the 1981 ICN/UCLA Symposium on DevelopmentBiology Using Purified Genes held in Keystone, Colo. on Mar. 15-20 1981,volume xxiii, 19891, pp647-658, Academic Press; editor Donald E Brown etal, or even enzyme labelled by the method of A D B Malcolm et al,Abstracts of the 604th Biochemical Society Meeting, Cambridge, En-land(meeting of Jul. 1 1983). The aforementioned protein HUBC4 may also bedetermined by the use of antibodies which may be polyclonal but arepreferably monoclonal, raised to a polypeptide sequence coded for by atleast a portion of the aforementioned genomic DNA sequence orcorresponding RNA sequence. The antibody may thus bind to the proteinencoded by the aforementioned genomic DNA sequence or corresponding RNAsequence or bind to any fragment of the protein. In addition, antibodyfragments, as aforedescribed, may also be used for this purpose.

The said antibodies of the present invention may, if desired, carry alabel or marker component, for example, as hereinbefore described inrelation to the polynucleotide probes of the present invention. Thus theantibodies may, for example, carry a fluorescent marker. It is not,however, necessary that the antibodies of the present invention carry alabel or marker component. Thus, for example, the antibodies of thepresent invention may be identified by a second antibody which is anantibody to antibodies of the present invention, for example, goatanti-mouse immunoglobulin. In this instance, the second antibody willhave a labelled or marker component.

For the diagnosis of a predisposition to Alzheimer's Disease, theinvention may be conveniently practised in the following fashion. mRNAis isolated from the peripheral white blood cells of the subject forinvestigation by standard techniques. This is copied into singlestranded cDNA using oligo-dT as a primer and, for example, reversetranscriptase. The single stranded cDNA may be converted into a doublestranded form arid cloned in a plasmid. bacteriophage or cosmid vector.Clones containing the HUBC4 sequences may be identified usingpolynucleotide probes from within the HUBC4 gene sequence as definedabove. The cDNA sequence in such clones may be determined using standardtechniques, for example, using an Applied Biosystems Model 373A-01 DNASequencer. The sequence thus obtained can be compared with the normalsequence provided for HUBC4 herein. In this way any mutations occurringin the HUBC4 mRNA in the individual under investigation will bedetected. It will be appreciated by the skilled man that alternativemethods of analysis of mRNA in patient samples can be used in thepractice of the invention. For example, the technique of RT-PCR (ReverseTranscription Polymerase Chain Reaction) may be applied. In this case,one of both of the oliogonucleotides used in a PCR reaction will bederived from within the HUBC4 coding sequences provided herein (see FIG.13).

An alternative method of putting the invention into practice wouldinvolve the analysis of DNA from an individual. Such analysis isconveniently performed by PCR amplification of the HUBC4 genes usingpolynucleotides or oligonucleotides capable of hybridising to anyportion of the HUBC4 cDNAs or to any portion of the HUBC4 genes or toany portion of the DNA sequence contained within the Yeast ArtificialChromosomes LMM-YAC1, LMM-YAC2, LMM-YAC3 or LMM-YAC4 or the cosmidsLMM-COS1, LMM-COS2 and LMM-COS3. PCR products thus obtained may beconveniently sequenced directly by methods well known in the art toestablish differences between sequences in an individual and the normalsequence of the HUBC4 genes.

The practice of the invention can further be carried out by theinsertion of HUBC4 cDNA or gene sequences into a gene targeting vectordesigned to allow homologous recombination between exogenous targetingDNA and endogenous target gene, for example, in mouse embryo stem cellsin culture. Homologous recombination may be performed using variants ofthe human or mouse UBC4 gene with either the normal or specific mutantsequences. The technique of gene knockout may be employed by performinghomologous recombination between the UBC gene in mouse or other animalspecies and in particular in embryonic stem cells derived therefrom,with a modified UBC4 gene of the type disclosed herein where the codingsequence of such UBC4 gene has been deliberately disrupted by theinsertion of exogenous nucleic acid sequence. Such exogenous nucleicacid sequence may, for example, be itself capable of encoding a protein.For example, a gene encoding a protein confirming neomycin resistancecan be inserted into UBC4 coding sequences so that after homologousrecombination UBC function is destroyed whilst the transfected cell linebecomes capable of growth in large quantities of neomycin or G418. Avariety of other techniques well known in the art for production oftransgenic animals are equally applicable in the practice of the presentinvention. This will include the direct injection of UBC4 nucleic acidsequences into pronuclei or to introducing such mutations bytransfection of embryonic stem cells, reintroduction into blastocystsand the breeding of chimeric animals.

The method of the invention may also be performed by the use ofantibodies recognising epitopes within the HUBC4 proteins derived asdiscussed above. Individuals who have HUBC4 alleles which result in thegeneration of a truncated form of the protein may be identified byWestern blot analysis of proteins from, for example, their peripheralblood white cells or in cells obtained by lumbar puncture from thecerebrospinal fluid. Heterozygotes for HUBC4 mutations (and individualswho are hence at risk of AD given that the disease is autosomaldominant) will display proteins of two sizes when analysed by Westernblots with the antibodies of the present invention. Such individualswill produce a normal protein plus a shorter truncated version. In asimilar fashion it will be possible to detect splice variants of theHUBC4 protein which may in fact produce variant HUBC4s which are largerthan normal.

Treatment of a Degenerative Disease

The DNA and/or protein sequence stricture, or any part thereof shown inFIGS. 1, 2 or 14 can be used for the treatment of the disease insofar asnon-mutant versions of HUBC4 can be manufactured using conventionaltechniques and subsequently delivered, again using conventional deliverymeans (see section above "Liposome mediated delivery of nucleic acidsand proteins to eukaryotic cells), to a target site with a view topreventing the onset of a degenerative disease such as Alzheimer'sDisease, or alternatively, mitigating the effects of a degenerativedisease such as Alzheimer's Disease or Down's Syndrome.

The DNA sequence structure shown in FIGS. 1, 2 14, or any part thereofmay be recombinantly introduced into a host cell (see section above"Expression of HUBC4 in prokaryotic cells") and subsequently expressedfor the purpose of supplying the corresponding protein. This protein maybe packaged within a liposome and delivered to the CNS directly byinjection or the like.

Alternatively, conventional recombinant vectors may be used to carry thegenetic sequence structure, or part thereof, shown in FIGS. 1, 2 or 14to the target site and also to express said sequence structure so as toprovide a non-mutant form of ubiquitin conjugating enzyme at the targetsite.

The Development of Therapeutics

Using conventional techniques non-human transgenic animals can beproduced, which animals would be provided with mutant forms of the genesand corresponding proteins shown in FIGS. 1, 2, 3, 4 and 14. Inaddition, these animals may be modified such that these genes, or apart, are absent or not expressed. Such animals will have apredisposition towards a degenerative disease such as Alzheimer'sDisease or cancer and will be useful in subsequent investigations forthe development and testing of therapeutic agents active against adegenerative disease such as Alzheimer's Disease or cancer.

REFERENCES

1. Proc. Natl. Acad. Sci. USA Vol 91 pp 8797-8801 (1994) Identificationof a human ubiquitin-conjugating enzyme that mediates theE6-AP-dependent ubiquitination of p53.

2. Journal of Biological Chemistry Vol 269 No. 13 pp 9582-9589 (1994)Degradation of the Tumour Suppressor Protein p53 by theUbiquitin-mediated Proteolytic System Requires a Novel Species ofUbiquitin-Carrier Protein, E2.

3. Cell Vol 75, 495-505 (November 1993) The HPV-16 E6 and E6-AP ComplexFunctions as a Ubiquitin-Protein Ligase in the Ubiquitination of p53.

4. Journal of Biological Chemistry Vol 269 No. 13 pp 9574-9581 (1994)Purification and Characterisation of a Novel Species ofUbiquitin-Carrier Protein E2, That Is Involved in Degradation ofNon-"N-end Rule" Protein Substrates.

5. Proc. Natl. Acad. Sci. USA Vol. 90 pp 10484-10488.

6. J. Biolog Chem Vol. 269 pp 8797-8802.

7. Genomics 12 447-453.

8. Rogers S. et al Science 234 364-368 1986.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 21    - (2) INFORMATION FOR SEQ ID NO: 1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1000 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - CCAGCCTCGC CAATATGGTG AAACCCGTTT CTACTAAAAA TATTTAAAAA AT - #TAGCCAGG      60    - CGTGGTGGCA ATCACCTGTA ATCTCAGTTA CTCGGGAGGC TGAGACAGGA GA - #ATTGCTTG     120    - AACTCAAGAG GCAGAGGTTG CAGTGAGCCA AGATTGAGCC ACTACACCCC AG - #CCTGGGTA     180    - ACACAGGGAG ACTCCATCTC AAAAATAAAA TAAAATAAAA TAAACAGGAG GA - #AGGAGCAG     240    - CACCAAATCC AAGATGGCGG CCAGCAGGAG GCTGATGAAG GAGCTTGAAG AA - #ATCCGCAA     300    - ATGTGGGATG AAAAACTTCT GTAACATCCA GGTTGATGAA GCTAATTTAT TG - #ACTTGGCA     360    - AGGGCTTATT GTTCCTGACA ACCCTCCATA TGATAAGGGG GCCTTCAGAA TA - #GAAATCAA     420    - CTTTCCAGCA GAGTACCCAT TCAAACCACC GAAGATCACA TTTAAAACAA AG - #ATCTATCA     480    - CCCAAACATC GACGAAAAGG GGCAGGTCTG TCTGCCAGTA ATTAGTGCTG AA - #AACTGGAA     540    - GCCAGCAACC AAAACCGACC AAGTAATCCA GTCCCTCATA GCACTGGTGA AT - #GACCCCCA     600    - GCCCAAGCAC CCGCTTCGGG CTGACCTAGC TGAAGAATAC TCTAAGGACC GT - #AAAAAATT     660    - CTGTAAGAAT GCTGAAGAGT TTACAAAGAA ATATGGGGAA AAGCGACCTG TG - #GACTAAAA     720    - TCTGCCACGA TTGGTTCCAG CAAGTGTGAG CAGAGACCCC GTGCAGTGCA TT - #CAGACACC     780    - CCGCAAAGCA GGACTCTGTG GAAATTGACA CGTGCCACCG CCTGGCGTTC GC - #TTGTGGCA     840    - GTTACTAACT TTCTACAGTT TTCTTAATCA AAAGTGGTCT AGGTAACCTG TA - #AAGAAAGG     900    - ATTAAAAATT TAAGATGTTC TAGTTCTGCT CTCTTTGTTT TAAAAATCAC TG - #CTTCAATC     960    #  1000            AAAA ACAATAAAAA GTGTTGATGA    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 154 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (ix) FEATURE:              (A) NAME/KEY: Protein              (B) LOCATION:1..154    #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Met Ala Ala Ser Arg Arg Leu Met Lys Glu Le - #u Glu Glu Ile Arg Lys    #                15    - Cys Gly Met Lys Asn Phe Cys Asn Ile Gln Va - #l Asp Glu Ala Asn Leu    #            30    - Leu Thr Trp Gln Gly Leu Ile Val Pro Asp As - #n Pro Pro Tyr Asp Lys    #        45    - Gly Ala Phe Arg Ile Glu Ile Asn Phe Pro Al - #a Glu Tyr Pro Phe Lys    #    60    - Pro Pro Lys Ile Thr Phe Lys Thr Lys Ile Ty - #r His Pro Asn Ile Asp    #80    - Glu Lys Gly Gln Val Cys Leu Pro Val Ile Se - #r Ala Glu Asn Trp Lys    #                95    - Pro Ala Thr Lys Thr Asp Gln Val Ile Gln Se - #r Leu Ile Ala Leu Val    #           110    - Asn Asp Pro Gln Pro Lys His Pro Leu Arg Al - #a Asp Leu Ala Glu Glu    #       125    - Tyr Ser Lys Asp Arg Lys Lys Phe Cys Lys As - #n Ala Glu Glu Phe Thr    #   140    - Lys Lys Tyr Gly Glu Lys Arg Pro Val Asp    145                 1 - #50    - (2) INFORMATION FOR SEQ ID NO: 3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 683 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - AGCACCAAAT CCAAGATGGC GGCCAGCAGG AGGCTGATGA AGGAGCTTGA AG - #AAATCCGC      60    - AAATGTGGGA TGAAAAACTT CCGTAACATC CAGGTTGATG AAGCTAATTT AT - #TGACTTGG     120    - CAAGGGCTTA TTGTTCCTGA CAACCCTCCA TATGATAAGG GAGCCTTCAG AA - #TCGAAATC     180    - AACTTTCCAG CAGAGTACCC ATTCAAACCA CCGAAGATCA CATTTAAAAC AA - #AGATCTAT     240    - CACCCAAACA TCGACGAAAA GGGGCAGGTC TGTCTGCCAG TAATTAGTGC CG - #AAAACTGG     300    - AAGCCAGCAA CCAAAACCGA CCAAGTAATC CAGTCCCTCA TAGCACTGGT GA - #ATGACCCC     360    - CAGCCTGAGC ACCCGCTTCG GGCTGACCTA GCTGAAGAAT ACTCTAAGGA CC - #GTAAAAAA     420    - TTCTGTAAGA ATGCTGAAGA GTTTACAAAG AAATATGGGG AAAAGCGACC TG - #TGGACTAA     480    - AATCTGCCAC GATTGGTTCC AGCAAGTGTG AGCAGAGACC CCGTGCAGTG CA - #TTCAGACA     540    - CCCCGCAAAG CAGGACTCTG TGGAAATTGA CACGTGCCAC CGCCTGGCGT TC - #GCTTGTGG     600    - CAGTTACTAA CTTTCTACAG TTTTCTTAAT CAAAAGTGGT CTAGGTAACC TG - #TAAAGAAA     660    #               683ATGT TCT    - (2) INFORMATION FOR SEQ ID NO: 4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 659 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - CAGAATTTGG ATAAATAGGA GGCAGATTTG GCTTAAAAGC ACATTAGCTG TA - #AATCAGTT      60    - GTAAAGCCAG AGTTTTGTTC CCGGATTAGC TGCCTCTTGC CTGTGCCATT TC - #TGAGACTG     120    - TGTTAACCCC CCATGAATTG TCCTTCTCTT GGCAGTAATC CAGTCCCTCA TA - #GCACTGGT     180    - GAATGACCCC CAGCCTGAGC ACCCGCTTCG GGCTGACCTA GCTGAAGAAT AC - #TCTAAGGA     240    - CCGTAAAAAA TTCTGTAAGA ATGCTGAAGA GTTTACAAAG AAATATGGGG AA - #AAGCGACC     300    - TGTGGACTAA AATCTGCCAC GATTGGTTCC AGCAAGTGTG AGCAGAGACC CC - #GTGCAGTG     360    - CATTCAGACA CCCCGCAAAG CAGGACTCTG TGGAAATTGA CACGTGCCAC CG - #CCTGGCGT     420    - TCGCTTGTGG CAGTTACTAA CTTTCTACAG TTTTCTTAAT CAAAAGTGGT CT - #AGGTAACC     480    - TGTAAAGAAA GGATTAAAAA TTTAAGATGT TCTAGTTCTG CTCTCTTTGT TT - #TAAAAATG     540    - ACTGCTTCAA TCTACTTCAA AAGAATGGTG TTTCTTTTCT TGTCCAATTT TA - #TCCAAAAT     600    - CTTCAAGTTA CATTTAACCC ATAAGGTTTA AAAAAAAGGA AAAAAAACGG TT - #GTGGTTC     659    - (2) INFORMATION FOR SEQ ID NO: 5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 154 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Met Ala Ala Ser Arg Arg Leu Met Lys Glu Le - #u Glu Glu Ile Arg Lys    #                15    - Cys Gly Met Lys Asn Phe Arg Asn Ile Gln Va - #l Asp Glu Ala Asn Leu    #            30    - Leu Thr Trp Gln Gly Leu Ile Val Pro Asp As - #n Pro Pro Tyr Asp Lys    #        45    - Gly Ala Phe Arg Ile Glu Ile Asn Phe Pro Al - #a Glu Tyr Pro Phe Lys    #    60    - Pro Pro Lys Ile Thr Phe Lys Thr Lys Ile Ty - #r His Pro Asn Ile Asp    #80    - Glu Lys Gly Gln Val Cys Leu Pro Val Ile Se - #r Ala Glu Asn Trp Lys    #                95    - Pro Ala Thr Lys Thr Asp Gln Val Ile Gln Se - #r Leu Ile Ala Leu Val    #           110    - Asn Asp Pro Gln Pro Glu His Pro Leu Arg Al - #a Asp Leu Ala Glu Glu    #       125    - Tyr Ser Lys Asp Arg Lys Lys Phe Cys Lys As - #n Ala Glu Glu Phe Thr    #   140    - Lys Lys Tyr Gly Glu Lys Arg Pro Val Asp    145                 1 - #50    - (2) INFORMATION FOR SEQ ID NO: 6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 464 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - GAAGAAATAT GCAATTGTGG AATGAAAAAC TTCCGTAACT TCCAGGTAGA TG - #GAGCTAAT      60    - TTATTGACTT GGCAAGGGCT TATTGTTCCT GACAACCCTC CATATAAGGG GG - #CCTTCAGA     120    - ATCGAAATCA ACTTTCCAGC AGAGCACCCA TTCAAACCAC CGAAGAGCAC AC - #TTAAAGAT     180    - CTGTCACCCA AATGTCCACT AAAAGGGGCA GGTCTCTCTG CCAGTAAATT AG - #TGCTGAAA     240    - ACTGGAAGCC AGCAACCAAA ACTGACCAAG TAATCCAGTC CCTCACAGCA CT - #GGTGAATG     300    - ACCCCCAGCC TGAGCATCCA CTTCAGGCTG ACCTAGCTGA ATAATACTCT AA - #GGACTGTA     360    - AATATTTCTG TAAGAATGCT GAAGTTTACA GAGAAATAGG GGGAAAAGCG AC - #TTGTAGAC     420    #464               TGGC TCCAGTAAGT GTGAGCAGAG ACCC    - (2) INFORMATION FOR SEQ ID NO: 7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 90 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (ix) FEATURE:              (A) NAME/KEY: Protein              (B) LOCATION:1..154    #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Glu Glu Ile Cys Asn Cys Gly Met Lys Asn Ph - #e Arg Asn Phe Gln Val    #                15    - Asp Gly Ala Asn Leu Leu Thr Trp Gln Gly Le - #u Ile Val Pro Asp Asn    #            30    - Pro Pro Tyr Lys Gly Ala Phe Arg Ile Glu Il - #e Asn Phe Pro Ala Glu    #        45    - His Pro Phe Lys Pro Pro Lys Ser Thr Leu Ly - #s Asp Leu Ser Pro Lys    #    60    - Cys Pro Leu Lys Gly Ala Gly Leu Ser Ala Se - #r Lys Leu Val Leu Lys    #80    - Thr Gly Ser Gln Gln Pro Lys Leu Thr Lys    #                90    - (2) INFORMATION FOR SEQ ID NO: 8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 361 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - AACTGGAAGC CAGCCACCAA GACTGTCCAA GTAATCCAGT CCCTCATAGC AC - #TGGTGAAT      60    - GACCCCCAGC CTGAGCACCC ACTCCGGGCT GACCTAGCTG AAGAATACTC TA - #AGGACCGT     120    - AAAAAATTCT GTAAGAATGC TGAAGAGTTT ACAAAGAAAT ATGGGGAAAA GC - #GACCTGTG     180    - GACTAAAATC TGCCACGATT GGTTCCAGCA AGTGTGAGCA GAGACCCCGA GC - #AGTGCATT     240    - CAGACACCCC GCAAAGCAGG ACTCTGTGGA AATTGACACG TGCCACCAAC TG - #GCGTCCGC     300    - TTGTGGCAGT TACTAACTTT CTACAGTTTT CTTAATCAAA AGTGGTCTAG GT - #AACCTGTA     360    #              361    - (2) INFORMATION FOR SEQ ID NO: 9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 61 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Asn Trp Lys Pro Ala Thr Lys Thr Val Gln Va - #l Ile Gln Ser Leu Ile    #                15    - Ala Leu Val Asn Asp Pro Gln Pro Glu His Pr - #o Leu Arg Ala Asp Leu    #            30    - Ala Glu Glu Tyr Ser Lys Asp Arg Lys Lys Ph - #e Cys Lys Asn Ala Glu    #        45    - Glu Phe Thr Lys Lys Tyr Gly Glu Lys Arg Pr - #o Val Asp    #    60    - (2) INFORMATION FOR SEQ ID NO: 10:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 18 nucleoti - #des              (B) TYPE: nucleic acids              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #  18              AG    - (2) INFORMATION FOR SEQ ID NO: 11:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 21 nucleoti - #des              (B) TYPE: nucleic acids              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #21                TGCT G    - (2) INFORMATION FOR SEQ ID NO: 12:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 21 nucleoti - #des              (B) TYPE: nucleic acids              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #21                AGAC C    - (2) INFORMATION FOR SEQ ID NO: 13:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 22 nucleoti - #des              (B) TYPE: nucleic acids              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #                 22ACC AC    - (2) INFORMATION FOR SEQ ID NO: 14:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 21 nucleoti - #des              (B) TYPE: nucleic acids              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #21                ACCG A    - (2) INFORMATION FOR SEQ ID NO: 15:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 18 nucleoti - #des              (B) TYPE: nucleic acids              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    #15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #  18              GC    - (2) INFORMATION FOR SEQ ID NO: 16:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 19 nucleoti - #des              (B) TYPE: nucleic acids              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    #16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 19               CTT    - (2) INFORMATION FOR SEQ ID NO: 17:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 19 nucleoti - #des              (B) TYPE: nucleic acids              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    #17:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 19               CTT    - (2) INFORMATION FOR SEQ ID NO: 18:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 18 nucleoti - #des              (B) TYPE: nucleic acids              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    #18:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #  18              GG    - (2) INFORMATION FOR SEQ ID NO: 19:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 19 nucleoti - #des              (B) TYPE: nucleic acids              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    #19:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 19               GCA    - (2) INFORMATION FOR SEQ ID NO: 20:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 21 nucleoti - #des              (B) TYPE: nucleic acids              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    #20:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #21                TGAT G    - (2) INFORMATION FOR SEQ ID NO: 21:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 18 nucleoti - #des              (B) TYPE: nucleic acids              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    #21:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #  18              CG    __________________________________________________________________________

We claim:
 1. An isolated nucleic acid molecule which encodes apolypeptide, said polypeptide having an amino acid sequence encoded by anucleic acid molecule having a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 1, 3, 4, 6 and
 8. 2. The isolated nucleicacid molecule according to claim 1 wherein said isolated nucleic acidmolecule is from a human.
 3. The isolated nucleic acid moleculeaccording to claim 1 wherein said isolated nucleic acid molecule is froma mouse.
 4. The isolated nucleic acid molecule according to claim 1wherein said nucleic acid molecule has a nucleotide sequence selectedfrom the group consisting of SEQ ID NOS: 1, 3, 4, 6 and
 8. 5. Theisolated nucleic acid molecule according to claim 1 wherein SEQ ID NO: 1comprises N at residue position 320, wherein N is V.
 6. The isolatednucleic acid molecule according to claim 1 wherein SEQ ID NO: 1comprises N at residue position 605, wherein N is B.
 7. The isolatednucleic acid molecule having a nucleotide sequence which is fullycomplementary to a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, 3, 4, 6 and
 8. 8. The isolated nucleic acidmolecule according to claim 7 further comprising a label.
 9. An isolatednucleic acid molecule, which hybridizes in Taq polymerase buffer at 65°C. to a nucleic acid molecule having a nucleotide sequence set forth inSEQ ID NO: 1 or 8, and wherein said isolated nucleic acid molecule has acomplementary sequence that hybridizes in Taq polymerase buffer at 65°C. to a nucleic acid molecule having a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO:
 21. 10. Theisolated nucleic acid molecule according to claim 9 further comprising alabel.
 11. An expression vector comprising an isolated nucleic acidmolecule according to claim 1, operatively linked to a promoter.
 12. Theexpression vector according to claim 11 further comprising an enhancer.13. The expression vector according to claim 11 wherein the isolatednucleic acid molecule comprises a nucleotide sequence selected from thegroup consisting of SEQ ID NOS: 1, 3, 4, 6 or
 8. 14. An isolated cellline or strain transformed with an expression vector according to claim11.
 15. A process for producing a recombinant cell which produces apolypeptide encoded by the isolated nucleic acid molecule according toclaim 1, wherein said process comprises transfecting a cell with saidisolated nucleic acid molecule and isolating said recombinant cell. 16.The process according to claim 15 wherein said cell has the capacity toexport said polypeptide.
 17. The process according to claim 15 whereinsaid cell is transfected with a mixture comprising a liposometransfection reagent and said isolated nucleic acid molecule.